Investigation Of Inhibiting A549 Cell Proliferation Mediated With RNA Interference Targeting To Gene Her2/neu

Lung cancer is the first cause of cancer death around the world.The cure rate is lower than 15%despite some developments in chemotherapeutic agents HER-2/neu gene also called erbB2,is a member of the epidermal growth factor receptor (EGFR)family and encodes a 185 kDa protein with tyrosine kinas activity and shows partial homology to the other epidermal growth factor receptors.The HER2/neu oncogene is over expressed in 30%of Non small cell lung cancer(NSCLC),and its signaling pathways play an important roles in tumor occurring,cancer cell growth, differentiation,metastasis,anti-apoptosis and resistance to chemotherapeutic agents.RNA interference(RNAi) is the phenomenon of sequence-specific,posttranscriptional gene silencing(PTGS) triggered by double-stranded RNA(dsRNA), which exists in extensive organisms such as Caenorhabditis elegans,plants, mammalian.Now RNAi has developed into a novel gene-blocking tool and used to knockdown specific gene expression efficiently in various species.Many great achievements have been made by its application includes screening new genes, functional genomics and gene therapy.However,there is elusive about the effects of depressing the expression of HER2/neu by RNAi on lung carcinoma cell line A549 cell proliferation.AimTo construct RNA interference vector to down-regulate HER2/neu gene and study the siRNA against HER2/neu effect on the cell cycle,cell proliferation of lung carcinoma cell line A549 in vitro.Methods1.Cell culture:lung carcinoma cell line A549 were cultured at 37℃/5%CO₂ in DMEM medium supplemented with 100IU/ml penicillin,10μg/ml streptomycin,and 10%heat-inactivated fetal calf serum(FCS).2.Construction of recombinant siRNA expression plasmids:â‘ Choose appropriate 19bp sequence on the transcript of target gene and blast to eliminate any target sequences with significant homology to other coding sequences.The oligonucleotides were synthesized by Shanghai Bioasia corporation.â‘¡Annealing of each pair of oligonucleotidesâ‘¢Double digestion of vector by endonucleaseâ‘£Ligation of annealed oligonucleotide inserts to linearized vector.⑤Transformation of DH5α⑥Identification of recombinant plasmids by sequencing.3.Cell Transfection:â‘ Optimizing the transfection condition according to manufacture's introductions,choosing the best ratio of FuGene6 to plasmid DNA.â‘¡Plate the lung carcinoma cell line A549 one day before the transfection experiment.When cells were 60-80%confluent,transfected cells according to the optimal condition.4.EGFP Reporter Assay:lung carcinoma cell line A549 were transfected by pEGFP-N2 and siEGFP(target to EGFP) plasmid.The transfected cell were observed by fluorescence microscope.5.Western blotting:three days after transfection,cells were washed with PBS and collected by scraping.They were lysed in ice-cold RIPA buffer and centrifuged. The supernatant was used for protein concentration determination by BCA-200 method.The proteins were resolved on 10%SDS-polyacrylamide gels,transferred onto nitrocellulose membranes,and incubated with the appropriate antibodies.The peroxidase-based detection was performed with Chemiluminescence Reagent(Pierce) according to the manufacturer's instructions.6.RT-PCR:Total RNA was prepared from A549 cells 48h after transfection using the Tripure reagent according to the manufacturer's protocol.RT-PCR amplification products were resolved by 1%agarose gel.7.Cell cycle analysis and growth rate:â‘ A549 cells were seeded in 6.0cm dishes with DMEM medium free of serum.The cells were synchronized in cell cycle 24h later.Then the cells were transfected with psilence 3.1-HER2 or psilence 3.1-Control vector with FuGene6 reagent.The cells were trypsinized 48h after transfection and washed by PBS for three times.FCM analysis were applied to measure cell cycle;â‘¡The cells were seeded in 24 well plates and transfected with psilence 3.1-HER2 or psilence 3.1-Control vector by using FuGene 6.On different time points after transfection,MTT method were applied to measure cell growth.All samples were done in tripicate and every sample counted was repeated at least 3 times.Results1.The recombinant siRNA expression plamids were successully constructed based on pSilencer3.1H1 vector targeted to HER-2/neu.2.Plasmid-based siRNA expression could knockdown the expression of exogenous reporter genes.After being transfected with siEGFP plasmids,the EGFP overexpression lung carcinoma cell line A549 shown decreased EGFP expression rate.3.Plasmid based siRNA expression could inhibit endogenous target genes. Several repeated experiments show that psilence 3.1-HER2 transfection can specially inhibit HER-2/neu mRNA and protein expression.4.FCM results showed that cells transfected with psilence 3.1-HER2 increased accumulation of cells in G₀/G₁ phase with a decrease in the percentage of cells in S-phase;Also,MTT results showed the growth curve of cells exhibited slower proliferation after the transfection of psilence 3.1-HER2.Conclusion1.human H1 promoter driven siRNA expression plasmids could be used in lung carcinoma cell line A549 to knockdown endogenous or exogenous gene expression.2.psilence 3.1-HER2 can knockdown her2/neu gene specially and efficiently; transfection of psilence 3.1-HER2 contributes to depressing effect of A549 cell proliferation.The data described here provided a proof of concept that vector-based RNAi targeting to her2/neu gene could be used therapeutically for lung cancer.